Low-intensity two-dimensional imaging of fluorescence lifetimes in living cells

The use of a time- and space-correlated single-photon counting detector enables us to perform fluorescence lifetime imaging microscopy in living cells with a temporal resolution of less than 100 ps and a spatial resolution of 500 nm. Two-dimensional (2D) maps of the fluorescence lifetimes and the corresponding prefactors are extracted by the use of a fitting program based on the Levenberg–Marquardt algorithm (Globals Unlimited). We applied this technique to extract 2D maps of protein localization in multilabeled living cells and to study protein–protein interaction by fluorescence resonance energy transfer.